The progress of science in genetics, genomics and biotechnology has been spectacular ever since Watson and Crick published the double helix structure of DNA in 1953 and the molecular biology era was thus inaugurated. One of the most extended analytical applications of DNA consists of detecting other DNA or RNA molecules with specific complementary sequences, given the binding specificity between nucleotides through their nucleotide bases, what is known as the Watson-Crick parity rule; adenine (A) with thymine (T), and guanine (G) with cytosine (C). Thus, a certain sequence formed by a certain number of nucleotides becomes unique inside the genome of one or more species, and said sequence can thus be used to detect and characterize the presence of the organism having it. Given the current technological development, it is even possible to detect the presence of an organism, strain or variant differing in only one nucleotide with respect to other variants, which allows distinguishing between two mutants of one and the same microorganism, for example.
The development of the technology of microarrays, also called chips or microchips (Southern et al., 1994; reviews in Nature Genetics 21, supplement, 1999; Harris, 2005), according to which thousands of molecular probes, mainly nucleic acids or proteins, can be covalently fixed to a solid support (glass, nitrocellulose, nylon, etc.), has entailed an important progress in the biotechnology field. Gene expression studies, nucleotide polymorphism (SNP) studies, microorganism typing and minisequencing can be carried out by means of DNA microarrays. This technique uses the experimental methodology developed by E. Southern (Southern, 1975), according to which nucleic acids (both long strands and short oligonucleotides) can be fixed to a solid support and form stable hybrids with their radioactively or fluorescently labeled complementary nucleic acids. The stability of the hybrids is determined by the degree of complementarity of the nucleotide sequences and by external factors such as the ionic strength of the medium, pH or temperature. (Parinov et al., 1996; Hacia, 1999; Relógio et al., 2002).
The use of molecules similar to natural nucleic acids for several applications has also been relevant in biotechnology. Within said molecules, the case of peptide nucleic acids (abbreviated as PNA), described for the first time by Nielsen et al. in 1991 (Nielsen et al., 1991), is particularly interesting. PNA consists of a polymer the backbone of which has a peptide nature, unlike the backbone of sugars and phosphates typical of natural nucleic acids (DNA and RNA). The PNA backbone is formed by N-(2-aminoethyl)glycine units joined by peptide bonds, it is achiral, electrically neutral and lacks phosphorus atoms (Egholm et al., 1992; Egholm et al., 1993). Purine (A and G) and pyrimidine (C and T) nucleotide bases are joined to the PNA backbone by means of methylene carbonyl bonds in a conformation such that they can interact exactly with the nucleotide bases of natural nucleic acids.
PNA is characterized by its capacity to hybridize stably and specifically with complementary DNA according to the Watson-Crick base parity rules (Egholm et al., 1993). In fact, single stranded PNAs (ssPNA) have greater affinity for complementary ssDNA than the ssDNA with a sequence identical to that of PNA. This is mainly due to the electrically neutral nature of PNA, preventing repulsion phenomena between strands present in DNA-DNA interaction (Nielsen et al., 1991; Wittung et al., 1994). The high affinity of PNA for DNA even allows hybridizing ssPNA to double stranded DNA (dsDNA) by means of a process called “strand invasion” (Demidov et al., 1995; Nielsen, 2001; Demidov et al., 2002) and allows using PNA probes to induce recombination and/or specific blocking of specific genes (Rogers et al., 2002). Furthermore, the interaction of PNA to DNA is very specific and for virtually all the base pairs which can be formed, the heat stability difference between correct and incorrect pairing is greater in a PNA-DNA duplex than in the DNA-DNA duplex (Egholm et al., 1993). Therefore, the temperature difference between that at which a complete pairing occurs and that in which one of the bases is unpaired is greater in the PNA-DNA case than in the DNA-DNA case. A biosensor based on immobilized PNA probes will thus be potentially more efficient than another biosensor based on DNA for detecting mutations and SNPs in a target nucleic acid molecule.
Given the structure of its artificial peptidomimetic backbone, PNA is not sensitive to the action of natural biodegrading enzymes such as DNases, RNases or proteases, therefore its biological stability is much greater than that of DNA or RNA. (Nielsen, 1999). Finally, the insensitivity of PNA to pH or ionic strength variations also makes it have a much greater chemical stability and offer greater experimental possibilities for its hybridization to different molecules in different medium compositions (Egholm et al., 1993; Kambhampati et al., 2001). Due to the foregoing, the exploitation of the physicochemical peculiarities of PNA for its use in systems for detecting and quantifying natural nucleic acids is evidently interesting.
Some of the physicochemical changes associated to PNA/DNA or PNA/RNA hybridization are obvious. One of them is the mass increase involved in this pairing; in this sense PNA/DNA(RNA) biosensors have been developed using a quartz crystal microbalance as a very sensitive instrument for detecting small mass changes occurring after the hybridization between complementary sequences (Wang et al., 1997) or mass spectroscopy in the MALDI-TOF modality (Griffin et al., 1997; Arlinghaus et al., 2003; Brandt et al., 2003).
Other systems for detecting the hybridization between a PNA probe and the DNA target have furthermore been developed which base the detection of hybridization on the appearance of a phosphorus signal, since this element is not present in the PNA strand but does form part of the target DNA (or RNA) strand backbone. This possibility has been shown by Arlinghaus et al. with the SIRIMP (Sputter-Initiated Resonance Ionization Microprobe Phosphorous Image) technique (Arlinghaus et al., 1997). Similar results have been obtained by the inventors of the present invention by using the X-ray photoemission spectroscopy (XPS) technique in PNA probes immobilized on planar plates with a gold surface, before and after the hybridization to complementary DNA targets. It has been observed by means of XPS that with hybridization (and after the corresponding washing in controlled conditions to prevent the non-specific binding of the target) there is an increase of between 2 and 4 times in the nitrogen signal (photoemission peak corresponding to N1s, normalized to the Au4f peak of the substrate), and the appearance of a phosphorus signal (P2p peak normalized to Au4f) which did not exist in PNA (Briones et al., 2004; Briones et al., 2005).
Conducting many of these assays in a homogeneous phase has a number of difficulties due to the fact that target DNA or RNA molecules are usually extremely diluted in natural samples, therefore the hybridization reaction is usually carried out on surfaces on which the PNA probe molecule has been previously immobilized. This represents a limitation of the assay because the amount of probe is limited to a layer of PNA molecules on the surface in question, with which the sample to be analyzed must necessarily be placed in contact in order to give a positive signal. The sensitivity and the detection limit of these techniques is therefore also limited.
At this point, the use of nanoparticles, and particularly magnetic nanoparticles, involves a very significant progress because a small amount of magnetic nanoparticles can be re-suspended in large sample volumes and can be subsequently recovered by means of applying an external magnetic field. It is thus possible to purify and/or pre-concentrate very minor and diluted amounts of the target DNA hybridizing specifically with the PNA immobilized on the nanoparticles, whereby the detection limit is reduced to a great extent. These types of systems allow determining the presence of specific DNA sequences of interest in situations in which an early detection thereof can be critical, for example for preventing harmful effects that the existence of the organism species or strains having said characteristic sequences may have. This fact has a great application in human and veterinary biomedicine, among others in the following aspects: i) detection of viral, bacterial, fungal or protozoan type pathogens; ii) characterization of mutations or genetic polymorphisms (SNPs) in said agents which can make them resistant to drugs or facilitate their escape from the immune system or to vaccines; iii) characterization of mutations or SNPs in human or animal genes, related to diseases or prone to them; iv) detection of specific tumor markers. This detection potential likewise has important applications in food and environmental control, in aspects including the following: i) detection of specific microorganisms, pathogens or contaminants; ii) detection of the presence of transgenic or genetically manipulated organisms (GMOs), being able to quantify if their presence is above the allowed limits. In all these cases, a considerable sample volume can be analyzed using hardly a few micrograms of nanoparticles in suspension, which are subsequently concentrated by means of an external magnetic field. It is thus possible to increase the sensitivity of the detection by several orders of magnitude.
Ferrofluids, which are stable ferromagnetic or ferromagnetic nanoparticle suspensions with a narrow particle size distribution, are particularly interesting for the analytical applications contemplated in this invention patent. These types of nanoparticles were initially obtained by means of mechanically grinding iron oxide samples with magnetic properties. However, this method, in addition to being expensive and slow, causes a high size distribution in the particles obtained. The alternative arose with co-precipitation methods, in which dissolved salts with the suitable ions (for example, Fe2+ and Fe3+ in a 1:2 ratio) are used and are taken to conditions in which said solution becomes unstable and the desired solid precipitates (in the same example, precipitation is achieved by taking the solution to 1M NaOH to boiling). These methods produce nanoparticles that are small enough to have superparamagnetism. This phenomenon involves that in one particle, due to its miniscule size, there is only one permanent magnetic domain, but with the capacity to rotate. Consequently, in a ferrofluid the magnetic moment of each nanoparticle is oriented at random and they cancel each other out, such that in the absence of an external field the fluid behaves as if it were not a magnetic solid. When these nanoparticles are subjected to a magnetic field, either by rotation inside the solution or by orientation of their magnetic field, they are oriented according to the external field. This causes a strong particle-particle attraction that is transmitted throughout the fluid. Although the possibility that the particles are concentrated through the application of an external magnetic field is obviously interesting, it is not desirable for the magnetic interactions to be so strong as to favor a collective orientation, and a possible aggregation and coagulation.
Therefore, to obtain a stable ferrofluid under a moderately strong magnetic field, i.e., that the force of attraction between particles is less than thermal energy associated to the particles, the latter must be very small. For the magnetic single-domain typical of superparamagnetism to appear, the upper limit of the particle size admitted at room temperature is about 3 nm for iron, and about 10 nm for Fe3O4 (magnetite) and for its oxidized form 3-Fe2O3 (maghemite). In the case of CoFe2O4 (cobalt ferrite), the particle size can reach up to 20 nm. Above these limits, particles that are too large can act as aggregation nuclei and grow, destabilizing the suspension. It is clear obvious that a small particle size and a narrow size distribution within the particle population are necessary. With these characteristics, ferrofluids are formed by stable phases of materials which can be moved or controlled by magnetic gradients. The three mentioned oxides, if they are synthesized in a suitable and coherent size, have a high applicability for these purposes.
The main direct method of obtaining magnetic nanoparticles of magnetite in solution, with a narrow size distribution, starts from iron salts (Massart, 1981). Magnetic nanoparticles can also be synthesized by means of vapor methods such as laser pyrolysis (Veintenillas et al., 1998), giving rise to very dispersed particles with a very small magnetic saturation, or the flame method (Urakawa et al., 1996) generating polydispersed particles. A silica matrix has also been used to obtain magnetic nanoparticles but the size distribution is too wide (del Monte et al., 1997).
Several synthesis methods for obtaining spherical magnetite nanoparticles in solution are currently known. According to one of said systems, a ferric hydroxide suspension is partially oxidized with different oxidizing agents. Spherical magnetite nanoparticles with a narrow size distribution for ranges selected from the limits 30 to 1100 nm have been obtained mixing FeSO4 with KOH in the presence of nitrate ion and taking the resulting gel to 90° C. for several hours (Sugimoto and Matijevic, 1980). However, the application of this method is limited because magnetite nanoparticles with diameters greater than 30 nm are no longer superparamagnetic.
Nevertheless, as has been indicated, the main method of obtaining spherical nanoparticles is the Massart method, consisting of aging a mixture of ferrous and ferric hydroxide solutions in an aqueous medium (Massart and Cabuil, 1987). Particles that are very homogeneous in size and chemical composition are obtained with a Fe2+/Fe3+ stoichiometry=0.5. It has furthermore been observed that, adjusting the pH and the ionic strength of the precipitation medium, the average particle size can be controlled in an order of magnitude within the nanometer range (between 1.6 and 12.5 nm) (Jolivet et al., 1983), such that the particle size decreases as the pH and the ionic strength increase. Both factors determine the isostatic change of the surface of the particles and consequently, the chemical composition of the surface.
In one of the descriptions for preparing this material, spherical magnetite particles between 8 and 14 nm were obtained by varying the nature of the base used in the precipitation (NH4OH, NaOH or KOH) and the temperature. To precipitate these particles, 50 ml of an aqueous 0.33 M FeCl2 and 0.66 M FeCl3 solution were added to 450 ml of a basic 1 M solution with strong stirring. A N2 gas flow was previously passed through the basic solution to ensure that the final precipitate was formed by magnetite only. The smallest particles were obtained by adding a 1 M KOH solution with 1% by weight of poly(vinyl alcohol) (PVA) to the mixture of iron salts at room temperature (Lee et al., 1996).
A method similar to the Massart method is used to synthesize CoFe2O4 nanoparticles, in which Fe2+ is changed for Co2+ and another one of the experimental conditions, such as the temperature for adding reagents to the basic medium, is changed. The process is similar to the magnetite method already mentioned above (Wagner et al., 2002).
The magnetic particles with a suitable size synthesized by the previous methods have an isoelectric point close to 7 for magnetite and of 9 in the case of cobalt ferrite, which makes them very unstable in aqueous solution at neutral pHs; certain aggregation is already observed at pH values±2 units of the isoelectric point, and they precipitate at pH values±1.5 units. For this reason, it is necessary to coat them with a compound stabilizing the suspension in water at pH values close to neutrality. This coating can be of different kinds: polymers, organic substances, or different metals or oxides. A great stability against aggregation is achieved in the event of coating particles with a silica layer, magnetic attractions and Van der Waals interactions occurring between said nanoparticles being reduced. A high resistance against heat treatments is furthermore provided (Tartaj et al., 2001). A problem involved with the silica coating is the low mechanical strength that said layer has inside a stirring tank. In addition, this silica coating could occur not on individual nanoparticles but on aggregates of about five nanoparticles (Philipse et al., 1994). On the contrary, the presence of said silica layer offers several advantages in addition to their stabilization at neutral pH, such as for example the possibility of modifying their surface by adding functional groups existing in silane type bond molecules. These molecules have a trialkoxysilane group at one of their ends, and a short chain with the relevant functional group (amino, mercapto, hydroxide, epoxide, etc) at the end of the chain comes out of the free bond of the silicon. Thus, instead of having the nanoparticles in a closed silica layer, they can be chemically modified to create the desired functionalized surface.
Thus, a modification with an outer gold layer has been carried out on silica nanoparticles (Oldenburg et al., 1998). To that end, the silica surface is silanized with 3-aminopropyltriethoxysilane, such that multiple amino groups are incorporated on the surface. Previously synthesized gold nanoparticles are immobilized on these groups and a subsequent growing step is carried out reducing gold (III) ions until desired, the gold layer on the silica being closed.
This outer metal layer (preferably gold or silver) adds many other properties to the silica-coated magnetic nanoparticles among which providing a singular optical property, the surface plasmon phenomenon, is worth emphasizing in relation to the present invention.
However, nanoparticles having all the previously described properties and the viability of their use as a support for detecting hybridization between complementary organic molecules and their application in the development of new tools or technological platforms in nanobiotechnology are not known.